Mod Spec® Service

quantitation of histone PTMs using mass spectrometry


Mod Spec® Service Overview

Mod Spec Service

Mod Spec® is a mass spectrometry method that is used to measure the relative abundance of > 60 different histone modifications. Mod Spec® is a great starting point to determine if epigenetic changes are occurring in response to disease or treatment. The large number of modifications monitored allows researchers to not only observe expected changes, but to discover unexpected changes, which can be even more informative.

Analyze histone modification changes in:

  • Response to epigenetic inhibitors
  • Normal vs diseased cells
  • Knock-out cells or animals
  • Inhibitor treated xenografts
  • Human biopsies

How Does Mod Spec® Work?

1. Histone Extraction: From 2-5 million cells or 25-100 mg tissue.

2. Propionylation: Mass spectrometry of proteins requires digestion of the proteins into small peptides before injection into the mass spectrometer. Propionylation, using propionic anhydride, blocks cleavage at lysines by trypsin, while allowing digestion at arginines to occur. The resulting peptides are of the appropriate size for mass spec analysis.

3. Trypsin Digestion: Histones are digested with trypsin.

4. Mass Spec: Samples are analyzed on a Triple-Stage Quadrupole Mass Spectrometer.

  • Q1: Peptides of the appropriate molecular weight are selected.
  • Q2: Peptides are further fragmented in the collision chamber.
  • Q3: Peptides fragments of interest are selected and analyzed by mass spectrometry.

5. Analysis: Fragments are analyzed and graphed to show changes between samples.

How does Mod Spec™ work?

Histone Modifications Detected by Mod Spec®

H1.4: K25UN
H1.4: K25AC
H1.4: K25ME1
H1.4: K25ME2
H1.4: K25ME3
^H2A: K5UN
^H2A: K5AC
^H2A: K9UN
^H2A: K9AC
^H2A: K36UN
^H2A: K36AC
^H2A1: K13UN
^H2A1: K13AC
^H2A1: K15UN
^H2A1: K15AC
^H2A3: K13UN
^H2A3: K13AC
^H2A3: K15UN
^H2A3: K15AC
*H3R2UN: K4ME1
*H3R2UN: K4ME2
*H3R2UN: K4ME3
H3: K9UN
H3: K9AC
H3: K9ME1
H3: K9ME2
H3: K9ME3
H3: K14UN
H3: K14AC
H3: K18UN
H3: K18AC
H3: K18ME1
H3: Q19UN
H3: Q19ME1
H3: K23UN
H3: K23AC
H3: K23ME1
H3: R42UN
H3: R42ME2
H3: R49UN
H3: R49ME2
H3: Q55UN
H3: Q55ME1
H3: K56UN
H3: K56AC
H3: K56ME1
H3: K64UN
H3: K64AC
H3: K79UN
H3: K79AC
H3: K79ME1
H3: K79ME2
H3: K79ME3
H3: K122UN
H3: K122AC
H3.1: K27UN
H3.1: K27AC
H3.1: K27ME1
H3.1: K27ME2
H3.1: K27ME3
H3.1: K36UN
H3.1: K36AC
H3.1: K36ME1
H3.1: K36ME2
H3.1: K36ME3
H3.3: K27UN
H3.3: K27AC
H3.3: K27ME1
H3.3: K27ME2
H3.3: K27ME3
H3.3: K36UN
H3.3: K36AC
H3.3: K36ME1
H3.3: K36ME2
H3.3: K36ME3
H4: K5UN
H4: K5AC
H4: K8UN
H4: K8AC
H4: K12UN
H4: K12AC
H4: K16UN
H4: K16AC
H4: K20UN
H4: K20AC
H4: K20ME1
H4: K20ME2
H4: K20ME3

^ Multiple H2A isoforms may contribute to the signal for modifications on H2A.

* H3R2me2 and H3K4me2/3 are mutually exclusive modifications. H3R2 methylation prevents H3K4 methylation. Therefore, H3K4 modifications  are reported only on the H3R2 unmodified peptide. For more information, see Nature. 2007 Oct 18; 449(7164):933-7.

H3.2 may contribute to signals for modifications labeled H3.1.

What our customers are saying about us:

"While working on the molecular mechanism of an epigenetic drug, we outsourced Mod Spec® to Active Motif in order to get a broader overview of the drug-induced changes of histone modifications. Overall, we were very pleased with the quality of the service, the kept timeline and last but not least, the fair price. We found surprising things that were not on our radar before."

Matthias Lauth, PhD
Phillips University
Marburg, Germany
View complete list of testimonials >


Mod Spec® Service Data

Mod Spec<sup>®</sup> data from control HeLa cells and HeLa cells treated for 7 days with 0.5uM GSK-126

Figure 1: Mod Spec® data from control HeLa cells and HeLa cells treated for 7 days with 0.5uM GSK-126.(Click image to enlarge)

GSK126 is an inhibitor that blocks the methytransferase activity of EZH2, resulting in global decreases in H3K27me2 and H3K27me3. A) A selection of H3 modifications are shown from Active Motif’s Mod Spec® assay that confirm significant decreases in H3K27 methylation with concomitant increases in acetylated and unmodified H3K27 (highlighted in yellow). Changes at H3K36 were also detected (highlighted in red). B) A selection of H4 modifications do not show significant changes in response to GSK126 treatment.

EZH2 inhibition results in decreased H2K27me3 levels

Figure 2: Mod Spec® data from control HEK293 cells and HEK293 cells treated for 6 hours with 5 mM sodium butyrate (NaB).

NaB is a general HDAC inhibitor and treatment is expected to increase histone acetylation levels. A) Mod Spec® data for a selection of acetylated and unmodified histone sites is shown. Acetylation is increased at all sites after treatment. Unmodified peptide is displayed for some sites and shows a concomitant decrease after NaB treatment. B) Methylation at various histone residues is not affected by NaB treatment.


Mod Spec® Service Publications

Search our database of customer publications that have used our Mod Spec® services.




Mod Spec® Service Documents




Mod Spec® Service Sample Submission Portal

Our online sample submission portal allows you to easily upload your service project samples and track your project status. Follow the sample submission instructions in the portal to ensure that all your samples arrive at Active Motif in the best possible condition and properly associated with your project.


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